Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors: In Vitro and In Vivo Post-grafting Safety Characterization

The critical requirements in developing clinical-grade human-induced pluripotent stem cells–derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable in vivo post-grafting differentiation, and acceptable safety profile. Here, we report on the use of manual-selection protocol for generating expandable and stable human NPCs from induced pluripotent stem cells. The hiPSCs were generated by the reprogramming of peripheral blood mononuclear cells with Sendai-virus (SeV) vector encoding Yamanaka factors. After induction of neural rosettes, morphologically defined NPC colonies were manually harvested, re-plated, and expanded for up to 20 passages. Established NPCs showed normal karyotype, expression of typical NPCs markers at the proliferative stage, and ability to generate functional, calcium oscillating GABAergic or glutamatergic neurons after in vitro differentiation. Grafted NPCs into the striatum or spinal cord of immunodeficient rats showed progressive maturation and expression of early and late human-specific neuronal and glial markers at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted brain or spinal cord samples. These data demonstrate the effective use of in vitro manual-selection protocol to generate safe and expandable NPCs from hiPSCs cells. This protocol has the potential to be used to generate GMP (Good Manufacturing Practice)-grade NPCs from hiPSCs for future clinical use.


Introduction
The discovery of human somatic cell reprogramminginduced pluripotent stem cells (hiPSCs) technology 1,2 provided remarkable progress in the potential development of patient-specific cell-replacement-based therapies. Besides, the generation of specific cell types from hiPSCs derived from patients with a defined genetic mutation proved to be a valuable platform for drug target discovery or creating human disease modeling both in vitro and in vivo [3][4][5][6] .
Several factors are being considered in the process of hiPSC generation particularly if generated cell lines or their lineage-committed derivatives are planned to be used in prospective human clinical trials. First is the source of somatic cells to be used in reprogramming to generate hiPSCs. The most frequently used cell types are skin fibroblasts harvested by biopsies 21 . The key, but relative, limitation in using fibroblasts for reprogramming is the invasive nature to obtain the biopsy material and the need for the initial in vitro expansion of collected fibroblasts before the reprogramming process can be initiated 22 . The use of peripheral blood mononuclear cells (PBMCs) has been recently used for reprogramming 23,24 . The primary advantage in using PBMCs is a relative simplicity in obtaining the blood samples and the ability to collect a sufficient number of cells from a single blood draw needed for reprogramming (ie, no need for cell expansion). Also, the collection can be repeated if additional samples are needed. Second, is the use of reprogramming vector(s). The majority of initial studies have employed integrating viral vectors, including retrovirus for reprogramming 1,25 . The major limitation in using integrating vectors for reprogramming is the risk of mutagenesis and resulting tumorigenicity after in vivo grafting [26][27][28] . More recently, the use of several non-integrating vectors to generate hiPSCs from adult somatic cells was reported 29 . One of the non-integrating reprogramming vectors, the Sendai-virus (SeV; a single-stranded RNA virus), has been demonstrated to reprogram somatic cells with high efficiency, low cytotoxicity, and minimal risk of being integrated into the host genome 30 . Using SeV (encoding Yamanaka factors: SOX2, c-MYC, Klf4, and OCT3/4), several research groups generated the hiPSCs by reprogramming human PBMCs 23,24,31,32 . SeVreprogrammed hiPSCs were further successfully used for the generation of lineage-committed cell line derivatives, including photoreceptor-like cells 33 , and cardiomyocytes 34 .
Here, we report on a successful generation of in vitro expandable SeV-hiPSCs-NPCs. Established NPCs show a long-term in vitro expandability while maintaining a stable karyotype, expression of neural precursor markers but with a lack of pluripotent markers. Upon transplantation into the striatum or spinal cord of immunodeficient rat grafted NPCs acquire a neuronal and glial phenotype with no detectable tumor formation for up to 6 months post-grafting. These data show that the NPCs manual-selection derivation protocol by using SeV-generated hiPSCs can successfully be used and considered for the future generation of clinical GMP-grade NPCs to be used in prospective clinical trials in the treatment of several neurodegenerative disorders, including spinal ischemic or traumatic injury, ALS, stroke, or brain trauma.

Development of hiPSC-NPCs
This study was approved by the University of California, San Diego (UCSD) Internal Review Board (IRB) (approval ID 101323).
Quantitative polymerase chain reaction assessment of residual SeV. RNAs were prepared from (1) non-infected PBMCs (negative control), (2) SeV-infected PBMCs (3 days post-SeV infection; positive control), and (3) established iPSC line (LPF11; passage 5). cDNAs were synthesized using 1,000 ng of RNAs with Qiagen Quantitect RT Kit (in 20 μl of reaction volume). Quantitative polymerase chain reaction (Qpcr) was performed with TaqMan probes against SeV (custom-made probe targeting the SeV genome located between the coding region of NP and P protein) and β-actin. cDNAs were synthesized from 6.25, 6.25, and 1.56 ng of RNA prepared from negative-control PBMCs, positive-control PBMCs (3 days post-SeV infection), and established iPSC line (LPF11; passage 5), respectively. cDNA of positive control was diluted 160-fold. Conditions of PCR reaction are as follows: 50°C for 2 min, 95°C for 10 min and 40 cycles of (95°C for 15 s and 60°C for 1 min).
NPCs isolation. Once rosettes-like structures were observed (typically 3-4 days after EBs plating), approximately 100 to 200 morphologically defined NPCs that appeared outside of each rosette were manually picked up by pipette tips and transferred in PLO/L-coating 48-well plates. Isolated NPCs were cultured in the same media as described above for the whole duration of cell expansion. Cells were repeatedly passaged over 10 times. To examine the genetic stability of generated NPCs, karyotype was examined at passage 12 (Cell Line Genetics, Madison, WI, USA).
Doubling time. The NPCs were cultured to passage 20, seeded at 2 × 10 5 cells per well in PLO/L-coated six-well plates. Three wells per day were then separately dissociated with Trypsin and collected in separate tubes to be counted using the Countess II automated cell counter (Thermo Fisher Scientific). The doubling time was calculated using the following equation:

Animal Studies
All animal studies were approved by the UCSD Institutional Animal care and Use Committee (Protocol No. S01193).

NPCs Grafting and Differentiation In Vivo
Intra-striatal cell transplantation. Adult immunodeficient rats (Crl:NIH-Foxn1 rnu ; Charles River; n = 8) (Charles River, Wilmington, MA, USA) were anesthetized with isoflurane (5% induction, 1.5%-2% maintenance) and placed in a stereotaxic apparatus to keep the head in a fixed position. After the scalp was shaved, a sagittal midline skin incision was performed to expose the skull. To permit an intra-parenchymal brain injection, a small borrow hole was drilled into the skull using a dental drill. The NPCs (30,000 cells/µl/injection) were injected into the striatum (stereotaxic X-Y-Z coordinates: bregma 0.5 mm, lateral to ± 3.0 mm, and 3.8, 5.0, and 6.2 mm depth: three injections delivered at each depth bilaterally) using a 34G needle interconnected with a digital microinjector (Tritech Research, San Diego, CA, USA).

Animal Perfusion-Fixation and Immunofluorescence Staining
At 2 months (n = 3) or 6 months (n = 5) after in vivo NPCs grafting rats were terminally anesthetized with pentobarbital (100 mg/kg; i.p.) and transcardially perfused with heparinized saline (100 ml) followed by 4% PFA in PBS. The brain and spinal cord tissues were then dissected, post-fixed with 4% PFA overnight at 4°C, and cryoprotected in 30% sucrose for a minimum of 5 days. Coronal brain or transverse spinal cord sections were then cut on a cryostat (20-30 µm thick) and stained by using a standard immunofluorescence (IF) protocol. Sections were first washed with 0.3 % Triton-100containing PBS (TX-PBS) 3 × 10 min and incubated with a blocking solution containing 5% normal donkey serum in 0.3% TX-PBS for 60 min. This was followed by incubation in primary antibodies (Table 1) in blocking solution overnight at 4°C. Sections were then washed 3 × 10 min in PBS and incubated with fluorophore-conjugated secondary antibodies diluted in 0.3% TX-PBS for 1 h. After 3× wash with PBS, nuclear staining was performed using DAPI solution (0.1 µg/ml) followed by three times rinses in PBS. Sections were then mounted on slides and covered with anti-fade mounting medium (ProLong; Thermo Fisher Scientific).
For in vitro cultured cells, NPCs plated on glass coverslips were fixed in 4% PFA for 20 min and washed 3 × 5 min in PBS. After blocking with 5% normal donkey serum for 30 min, cells were incubated with primary antibodies (Table 1) in blocking solution overnight at 4°C. After 3 × 5 min rinses in PBS, the cells were then incubated with fluorophore-conjugated secondary antibodies in 0.2% TX-PBS for 1 h followed by 3 × 5 min rinses in PBS. Nuclei were stained with DAPI solution (0.1µg/ml) for 3 min followed by 3 × 5min wash in PBS. Coverslips were then mounted on slides with anti-fade mounting medium (ProLong; Thermo Fisher Scientific). The images were acquired using fluorescence (Zeiss Axio Imager M2 Microscope(Zeiss, Oberkochen, Germany) with Stereo Investigator software (MBF Bioscience, Williston, VT, USA), and using a confocal microscope (Fluoview FV1000, Olympus) with Olympus FV10-ASW Viewer software.

Flow Cytometry
Flow cytometry analysis was performed by following the company's instruction with modification (BD Biosciences, San Jose, CA, USA). At first, the established NPCs were cultured in DMEM/F12 media supplemented with 10 ng/ml bFGF as described before. Once cells reached confluence, cells were dissociated with Accutase (cat. no. AT-104, Innovative Cell Technologies co., San Diego, CA, USA) and a single-cell suspension (1 × 10 6 cells per 1 ml sample)

Statistical Analysis
All data are reported as the mean ± SEM. An unpaired twotailed Student's t test was used for single comparisons of grafted cells at the 2-month and 6-month post-grafting. In each case, *P < 0.05 and **P < 0.01 were considered to be statistically significant. The GraphPad Prism software (version 6.0c; GraphPad Software Inc., San Diego, CA, USA) was used for all analyses.

Lack of Residual SeV in Established Pluripotent LPF11 Cells
Using qPCR, we first probe for the residual presence of SeV (targeting the SeV genome located between the coding region of NP and P protein) in established pluripotent LPF11 cells. Compared with positive control (PBMCs 3 days post-SeV infection), no residual SeV was detected ( Supplementary  Fig. 1).

Generation of NPCs from hiPSCs
To generate the hiPSC-NPCs, a previously developed protocol established to isolate the NPCs from human pluripotent ESs and porcine iPSCs was employed 36,37 . First, the peripheral mononuclear blood cells were isolated from human blood samples and reprogrammed by SeV vectors encoding octamer-binding transcription factor 4 (OCT4), (sex-determining region Y)-box2 (SOX2), Kruppel-like factor 4 (KLF4), and myelocytomatosis viral oncogene (L-MYC). After reprogramming, cells were cultured on matrix-coating dishes as a monolayer (Fig. 1A, B). Established pluripotent colonies (Fig. 1B) were manually dissociated and transferred into low attachment dishes to induced EBs (Fig. 1C). After 4 to 6 days, established EBs were transferred onto PLO/L pre-coated dishes and induced using NPCs induction media (see "Material and Methods" for details). Attached EBs gradually flattened in shape and the appearance of neural rosettes (NRs) was seen in about 4 to 7 days after EBs plating/induction (Fig. 1D).
Several cell clusters originating from NRs but localized at the edges of NRs were then manually picked and re-plated to P/L coating dishes. These cell populations were passaged several times until morphologically defined NPCs-like cell groups were identified. Colonies of NPCs-like cells were then manually harvested, expanded on P/L coated dishes (Fig. 1E), and used in all subsequent in vitro and in vivo grafting experiments (Fig. 1A).

Established hiPSCs-NPCs Differentiate Into Functional Neurons and Glial Cells In Vitro
A defining characteristic of multipotent NPCs is their ability to generate functional neurons and glial cells (astrocytes and oligodendrocytes) after differentiation. To probe for the multi-lineage potency of established NPCs (passages 11-15), cells were treated with differentiation media (containing 10 ng/ml BDNF and 10 ng/ml GDNF) for 2 months. Induced cells were analyzed by (1) IF using neuronal and glial markers, (2) in situ fluorescence hybridization to identify glutamatergic or GABAergic mRNA transcripts, and, (3) by calcium oscillation imaging to confirm the presence of action potential-generating neurons.
To study the ability of induced neurons to generate action, potential cells were loaded with Fluo-4 AM and spontaneous calcium oscillation measured using high-resolution  fluorescence microscopy. Numerous neurons with calcium oscillation patterns consistent with spiking neurons (ie, periodic, quasi-periodic, or chaotic spiking's, as well as bursting's activities) were seen (Fig. 3K).
Taken together, these data demonstrate that established NPCs have multi-lineage potential and can generate maturefunctional excitatory inhibitory neurons and glial cells (astrocytes and oligodendrocytes).

In Vivo Grafted NPCs Are Non-Tumorogenic and Differentiates Into Neurons and Glial Cells in the Striatum and Spinal Cord of the Immunodeficient Rat
To characterize the safety and differentiation potential of established NPCs, cells were grafted into striata or lumbar spinal cord in the adult immunodeficient rat. Animals were sacrificed at 2 or 6 months post-grafting and the presence of grafted cells analyzed by H&E staining or IF using humanspecific and non-specific antibodies (Fig. 1A).
IF analysis of NPCs-grafted striata and spinal cords at 2 months postgrafting showed a consistent presence of grafted human cells stained with a human-specific nuclear marker  Figs. 2A, B, G, H, and 3A, C, D, E).
IF analysis at 6 months post-grafting showed a more advanced stage of grafted NPCs maturation in both striatum and spinal cord. Staining with mature neuronal markers (hNSE and NeuN) showed a high number of intensely hNSEstained and NeuN-stained neurons throughout the grafts (Figs. 4B, C, E, G and 5C, D, E). An intense hSYN in the graft and often associated with peripherally projecting HO14+ human axons was also seen (Figs. 4C, D, and 5B). Co-staining with hSYN and VGAT or VGLUT1-3 showed the presence of both inhibitory (VGAT+) and excitatory (VGLUT1-3) puncta co-localizing with grafted neuronsderived hSYN+ terminals (Figs. 4F and 5F, H). Compared with 2 months post-grafting, the density of GFAP+ astrocytes was clearly increased in the graft as well as at the border of the graft with the host tissue (Figs. 4A, B, and 5A, C). In the same regions, a continuing presence of early glial marker vimentin was seen, suggesting an ongoing glial proliferation (Figs. 4G and 5E). Oligodendrocyte staining with Olig2 antibody (co-staining with hNUMA) showed the majority of double-stained grafted cells within the grafts (Figs. 4H and  5G). Hematoxylin and eosin (H&E) staining of striatal sections showed normally appearing mature neural grafts with no sign of hyper-cellularity (which would be indicative of tumor formation) (Fig. 4I-1, 2). Quantitative analysis of DCX-, NeuN-, vimentin-, hGFAP-, Olig2-, and Ki67-positive cells at 2 and 6 months is provided in Figs. 4J and 5J.

Discussion
Using SeV-reprogrammed hiPSCs, we demonstrate an effective generation of NPCs by using a manual-selection protocol. Established NPCs line can be expanded for over 20 passages while maintaining a stable karyotype, expression of neural precursor markers, and with no signs of spontaneous differentiation. No expression of pluripotent markers was seen in established NPCs. In vitro-induced NPCs show differentiation toward glial phenotype (oligodendrocytes and astrocytes) and excitatory and inhibitory neurons capable of generating spontaneous action potentials. Established NPCs grafted into striata or spinal cord of immunodeficient rats show robust engraftment and neuronal and glial differentiation at 2 or 6 months post-grafting. No tumor formation was seen in NPCs-grafted regions.

Strategies for Purification/Enrichment and Expansion of NPCs From Pluripotent Stem Cells
Several previous studies have demonstrated the successful use of fluorescence-activated cell sorting, by using several combinations of neural and/or neuronal cell surface markers to isolate/enrich the population of expandable neural/neuronal precursors [38][39][40][41][42][43] . While effective in generating welldefined NPC populations, these protocols require the use of GMP-grade antibodies and cell-sorting equipment(s). As such, routine use of the FACS protocol for clinical use is prohibitively expensive. In our previous study, we have developed and characterized a manual-selection protocol that permits a reliable morphologically defined selection of NPCs from human ES cell lines 37 . Our current study, which employed the same protocol, demonstrates that the NPCs can similarly be isolated from established hiPSCs. Comparably, as previously shown for ES-derived NPCs, a stable karyotype and lack of pluripotent markers were seen after longterm passaging of hiPSCs-NPCs. Mature neurons derived from established NPCs showed action potential-generating properties and expression of both excitatory and inhibitory neurotransmitter phenotype. These data are similar to other  studies that show the appearance of functional neurons after induction of porcine iPSCs-NPCs in vitro 36 or long-term maintained hiPSC-derived brain organoids 44,45 .

Selecting the Optimal In Vitro NPCs Culture Protocol to Generate Transplantable NPCs Cell Bank
One of the key determinants defining a successful and effective translation of cell-replacement-based therapies into clinical practice is the format on how the established NPCs cell lines are cultured, stored, and prepared for in vivo grafting. In general, two basic protocols are being employed to culture and expand NPCs whether derived from fetal central nervous system (CNS) tissue, ESs, or iPSC lines. First, the NPCs are cultured in the form of neurospheres 46 . For passage, the individual neurospheres are enzymatically or mechanically dissociated and then subcultured. While the in vitro culturing of established neurospheres is relatively straightforward and does not require any coating of tissue culture wells, there are three key limitations from the perspective of effective use of neurospheres in a clinical setting. (1) The freezing of established neurospheres is typically associated with a poor postfreezing cell/neurospheres recovery thus limiting the use of previously frozen neurospheres for routine clinical use. (2) Because the size of individual neurospheres varies significantly the required cell dosing to be delivered is difficult to control unless the filtering of prepared neurospheres culture is performed before grafting. (3) Finally, a relatively large size of neurospheres and tendency of aggregation prohibit the use of small diameter needles (~30G) for intra-parenchymal delivery. This can be of particular concern when the spinal delivery of cells (which requires smaller needles relative to brain injection cannulas) is desired [47][48][49] . Second, the NPCs can be cultured as a cell monolayer on previously coated tissue culture wells. For coating, a combination of PLO/L is typically used 50 . This culturing protocol has successfully been used to expand and bank clinical-grade NPCs derived from fetal tissue or ESs lines 49,51 . Our current protocol used a comparable approach with NPCs expanded as a monolayer on a previously P/L-coated surface. While relatively more laborious, this protocol has several specific advantages (compared with neurosphere technology) once considered for potential clinical application: (1) a consistent neural precursor cell morphology can be seen in established-proliferating NPCs, (2) minimal or no spontaneous differentiation is typically seen even with extensive passaging, (3) over 90% cell viability is usually seen in previously frozen cells after thawing, and (4) a relatively smaller injection needle (~30g) can be used without difficulties to deliver single-cell suspension into the brain or spinal cord in a large animal model(s) (such as pig or NHP) and in human 36,[47][48][49]52,53 . As such, the use of monolayer NPCs culturing and expansion is a preferable method in producing NPC cell bank(s) intended for clinical use.

Manually Selected and In Vitro-Expanded hiPSCs-NPCs Show Long-Term In Vivo Engraftment in the Absence of Tumor Formation
In our current study, in vivo grafted NPCs into striata and spinal cord of immunodeficient rats showed a robust neural differentiation with the presence of mature neurons and glial cells (oligodendrocytes and astrocytes). No tumor formation was seen at 2 or 6 months post-grafting. Mature grafted neurons showed extensive axodendritic sprouting and development of putative inhibitory and excitatory synaptic contacts with the host interneurons and CHAT+ α-motoneurons. Qualitatively, these data are very similar to the differentiation profile of human fetal spinal cord-, ES-, or iPSCsderived NPCs grafted spinally in naïve immunosuppressed adult pig or immunodeficient rat 53,54 , spinally injured rat or non-human primates 52,55-58 , or in rat with previous spinal ischemic injury 59,60 . Similarly as shown in previous studies, no excessive proliferation of grafted cells was seen and the only occasional presence of grafted Ki67+ cell was detected. Jointly these data demonstrate a high degree of phenotypic/ functional equivalency in established iPSCs-NPCs to fetal tissue-derived or ES-derived NPCs after long-term in vivo grafting. More recently, using SeV reprogrammed adult pig skin fibroblasts-derived iPSC-NPCs, we have demonstrated a comparable long-term (2-8 months) engraftment and functionality of NPCs after intra-striatal grafting in immunodeficient rat or after spinal grafting in syngeneic naive pig or allogeneic transiently immunosuppressed pig with previous spinal cord traumatic injury 36 .
In summary, we demonstrate a successful generation of expandable SeV-reprogrammed hiPSCs-NPCs line. Longterm expanded NPCs showed a stable karyogram and expression of typical neural precursor markers. In vitroinduced NPCs differentiate toward functional neurons, astrocytes, and oligodendrocytes. Long-term (6 months) in vivo-grafted iPSC-NPCs show a comparable engraftment/ differentiation profile and acceptable safety as previously reported for fetal tissue-derived or ES-derived clinicalgrade NPCs. Accordingly, this hiPSCs-NPCs cell line and/ or NPCs-selection protocol may represent an effective technology for generating clinical-grade NPCs to be used in the perspective human clinical trial(s) in the treatment of several spinal neurodegenerative disorders such as spinal ischemic/traumatic injury, ALS, or multiple sclerosis.